Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman's reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL−1. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC50 values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.
Aiming to identify new sources of bioactive secondary metabolites, we isolated 82
endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia
echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro
assays. The organic extracts from three isolates showed antibacterial activity
against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration
(MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC
64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL),
Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a
concentration of 20 μg/mL showed antitumour activities against human breast cancer
and human renal cancer cells, while two isolates showed anti-tumour activities
against human melanoma cancer cells. Six extracts were able to reduce the
proliferation of human peripheral blood mononuclear cells, indicating some degree of
selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania)
amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL.
The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected
to bioguided fractionation, which revealed beauvericin as the compound responsible
for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a
half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular
culture assay.
BACKGROUNDIn a screen of extracts from plants and fungi to detect antileishmanial
activity, we found that the ethyl acetate extract of the fungus
Nectria pseudotrichia, isolated from the tree
Caesalpinia echinata (Brazilwood), is a promising
source of bioactive compounds.OBJECTIVESThe aims of this study were to isolate and determine the chemical structures
of the compounds responsible for the antileishmanial activity of the organic
extract from N. pseudotrichia.METHODSCompounds were isolated by chromatographic fractionation using
semi-preparative high-performance liquid chromatography, and their chemical
structures were determined by analytical and spectral data and by comparison
with published data. The antileishmanial activity of the isolated compounds
was evaluated in intracellular amastigote forms of Leishmania
(Viannia) braziliensis expressing firefly luciferase as
reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian
cell lines by MTT assay.FINDINGSFractionation of the extract yielded seven compounds: 10-acetyl trichoderonic
acid A (1), 6′-acetoxy-piliformic acid (2), 5′,6′-dehydropiliformic acid
(3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and
cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first
time. Compounds 1, 2, and 5 were more active, with IC50 values of
21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and
THP-1 cells.MAIN CONCLUSIONS
N. pseudotrichia produces secondary metabolites that are
more toxic to intracellular amastigote forms of L. (V.)
braziliensis than to mammalian cells.
Paracoccidioidomycosis (PCM), a human mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis, is a serious public health problem in several countries of Latin America. In our search we found that the crude extract of the endophytic fungus UFMGCB 551 was able to inhibit several clinical strains of P. brasiliensis, and was also active in the bioautographic assay against Cladosporium sphaerospermum. The endophytic fungus UFMGCB 551 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae). The fungus was identified as Fusarium sp. based on its macro- and micro-morphology, and on the sequence of the internally transcribed spacer regions (ITS) of its rRNA gene. The chromatographic fractionation of the fungal extract was guided by the bioautographic assay to afford three known trichothecene mycotoxins: T2-toxin (1) and a mixture of 8-n-butyrylneosolaniol (2) and 8-isobutyrylsolaniol (3). The minimal inhibitory concentrations (MIC) of the these compounds against eleven clinical strains of P. brasiliensis were evaluated and found to be in the range between 75 and 640 nmol l(-1) for 1 and 160-640 nmol l(-1) for the mixture of 2 and 3.
Aim
This study aimed to isolate Pseudobrickellia brasiliensis endophytic bacteria and evaluate the production of hydrolytic enzymes and antibiotics by these bacterial strains. The study also measured the antibacterial activity of P. brasiliensis.
Methods and Results
Thirteen endophytic bacteria strains were isolated from stem and leaf fragments of P. brasiliensis. Extracellular enzyme production by the isolated endophytic bacteria was evaluated in an agar plate‐based assay. The highest protease production was achieved by Bacillus subtilis P4 in alkaline medium. Antimicrobial activity of endophytic bacteria and P. brasiliensis extracts was investigated using microbroth dilution. An MIC value of 1000 μg ml−1 against Pseudomonas aeruginosa was found for B. subtilis P3, B. subtilis P5, Pseudomonas sp. P8 and Pseudomonas sp. P12. Leaf extract of P. brasiliensis showed the highest antibacterial activity against P. aeruginosa, with an MIC value of 0·781 mg ml−1.
Conclusions
Pseudobrickellia brasiliensis is a source of bacterial endophytes, which can produce antibacterial compounds and enzymes. This work also demonstrated the antibacterial potential of P. brasiliensis.
Significance and Impact of the Study
This is the first study that revealed the antibacterial activity of P. brasiliensis and bioactive metabolite production by P. brasiliensis endophytic bacteria.
The bioassay-guided fractionation of the ethyl acetate extract of the fungus Cochliobolus sp. highlighted leishmanicidal activity and allowed for anhydrocochlioquinone A (ANDC-A) isolation. MS, 1D and 2D NMR spectra of this compound were in agreement with those published in the literature. ANDC-A exhibited leishmanicidal activity with EC value of 22.4 microgram/mL (44 mu M) and, when submitted to the microdilution assay against Gram-ositive and Gram-negative bacteria, showed a minimal inhibitory concentration against ATCC 25295 of 128 microgram/mL (248.7 mu M). It was also active against five human cancer cell lines, showing IC values from 5.4 to 20.3 mu M. ANDC-A demonstrated a differential selectivity for HL-60 (SI 5.5) and THP-1 (SI 4.3) cell lines in comparison with Vero cells and was more selective than cisplatin and doxorubicin against MCF-7 cell line in comparison with human peripheral blood mononuclear cells. ANDC-A was able to eradicate clonogenic tumour cells at concentrations of 20 and 50 mu M and induced apoptosis in all tumour cell lines at 20 mu M. These results suggest that ANDC-A might be used as a biochemical tool in the study of tumour cells biochemistry as well as an anticancer agent with durable effects on tumours.
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